The broad goal of our research program is to understand how neural circuit function depends on the intrinsic properties of component cells and synapses. The specific goal of this proposal is to determine how rod-mediated visual processing in the retina depends on the timing of signaling at rod bipolar (RB) cell synapses with postsynaptic AII amacrine cells. Transmission at the RB?AII synapse is shaped by intrinsic (e.g. synapse-specific dynamics of vesicle release and recycling) and extrinsic (e.g. inhibition of RBs by interneuron circuits) factors. This application comprises two specific aims that integrate synapse- and circuit-level analyses of RB?AII transmission. Specific Aim 1 tests the novel hypothesis that Ca2+ influx into the RB terminal regulates the strength and timing of transmission at the synapse by two calmodulin (CaM)-dependent mechanisms: one modulates membrane lipid composition by regulating a Ca2+-independent phospholipase A2 (iPLA2); the second affects a constitutively active brake on spontaneous release. Modulation of transmission is studied by electrophysiological experimentation, and temporal coding is examined in a retina in which RBs lack a fast mode of exocytosis: the complexin (clpx) 3 knockout. Specific Aim 2 determines the organization and physiological function of parallel inhibitory pathways converging on the RB. Scanning block face electron micrographic (SBEM) reconstruction identifies inhibitory amacrine cells (ACs) presynaptic to RBs. AC?RB synapses are studied functionally using various electrophysiological techniques: paired AC-RB recording, optogenetic stimulation of ACs, and optogenetic stimulation of bipolar cells-BCs-presynaptic to the ACs. Relevance to Public Health: Understanding how temporal coding is implemented by retinal synapses informs the design of retinal prosthetics and the study of animal models of human retinal diseases. A goal of vision research is the development of gene-based therapies for treating blindness caused by photoreceptor degeneration, and a promising therapy is the generation of light sensitivity in retinal interneurons by virally-mediated expression of channelrhodopsin-2 (ChR2), a light-gated cation channel. We will express ChR2 in interneurons to study synaptic interactions in retinal circuits and thereby generate critical information about the operating range of a retina in which ChR2 is the only light sensor. We address three goals of the Retinal Diseases Program in the National Plan for Eye and Vision Research: 1) determining potential therapeutic strategies for treatment of retinitis pigmentosa, 2) increasing understanding of post-photoreceptor adaptation (i.e. interactions between excitatory and inhibitory synapses), and 3) increasing understanding of how inter-cellular interactions in neural networks generate signals that are interpretable as visual images.